Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation.

Genomic instability at simple repeated sequences (SRS) is a landmark for some sporadic and hereditary cancers of the colon. We have identified several human tumour cell lines with up to 1,000-fold increases in mutation rates for endogenous microsatellite sequences, relative to normal cells or tumour cells without the mutator phenotype and show that they are very early events in tumorigenesis. Our in vivo and in vitro results show that the genomic instability persists after transformation and that microsatellite mutations accumulate as consecutive somatic slippage events of a single or a few repeated units. This mechanism may account for the repeat expansions in triplet hereditary diseases and the same defect in replication fidelity in non-polyposis colon cancer could also contribute to the non-mendelian anticipation in these diseases.

Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype.

Cancers of the microsatellite mutator phenotype (MMP) show exaggerated genomic instability at simple repeat sequences. More than 50 percent (21 out of 41) of human MMP+ colon adenocarcinomas examined were found to have frameshift mutations in a tract of eight deoxyguanosines [(G)8] within BAX, a gene that promotes apoptosis. These mutations were absent in MMP- tumors and were significantly less frequent in (G)8 repeats from other genes. Frameshift mutations were present in both BAX alleles in some MMP+ colon tumor cell lines and in primary tumors. These results suggest that inactivating BAX mutations are selected for during the progression of colorectal MMP+ tumors and that the wild-type BAX gene plays a suppressor role in a p53-independent pathway for colorectal carcinogenesis.

High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability.

The EPH/EFN family of receptor tyrosine kinases regulates cell adhesion and migration and has an important role in controlling cell positioning in the normal intestinal epithelium. Inactivation of EPHB2 has recently been shown to accelerate tumorigenesis in the colon and rectum, and we have previously demonstrated frequent frameshift mutations (41%) in an A9 coding microsatellite repeat in exon 17 of EPHB2 in colorectal tumors with microsatellite instability (MSI). In this study, we extended these analyses to extracolonic MSI cancers, and found frameshift EPHB2 mutations in 39% (25/64) of gastric tumors and 14% (8/56) of endometrial tumors. Regression analysis of these EPHB2 mutation data on the basis of our previously proposed statistical model identified EPHB2 as a selective target of frameshift mutations in MSI gastric cancers but not in MSI endometrial carcinomas. These results suggest a functional role for EPHB2 in gastric tumor progression, and emphasize the differences between the tumorigenic processes in MSI gastrointestinal and endometrial cancer.

Oncogene amplification during tumorigenesis of established rat fibroblasts reversibly transformed by activated human ras oncogenes.

Normal rat fibroblasts of the established cell line Rat 4 were cotransformed with activated human ras oncogenes and with a cloned chicken thymidine kinase (tk) gene. Linkage between tk and ras genes allowed the isolation of oncogene deletion revertants and of cell clones showing varying degrees of malignant phenotype. Southern and Northern experiments in concert with tumorigenicity assays show that the malignant transformation of these cells by mutant ras oncogenes is a gradual but reversible process that depends on the relative abundance of oncogene sequences and their corresponding transcripts. We also show that moderate amplification of a c-K-ras oncogene in these cells results in a clear increase in their tumorigenicity and that the mutant gene present in low copy numbers in cultured cells undergoes amplification in the corresponding in vivo induced tumors.

Genetic heterogeneity of the c-K-ras locus in colorectal adenomas but not in adenocarcinomas.

BACKGROUND: Previous molecular genetics studies of colorectal cancer have identified multiple mutations in the c-K-ras gene (also known as KRAS2) in all phases of its development. Because of technical difficulty, prior studies rarely focused attention on the detailed distribution of c-K-ras mutations in multiple regions of the same primary tumor specimen. However, with recent development of the selective UV radiation fractionation method, characterization of c-K-ras mutations in multiple regions of the same primary tumor specimen can be performed. PURPOSE: Our purpose was to describe how c-K-ras mutations were distributed among cells obtained from multiple regions of the same primary tumor in an attempt to describe differences between early and late colorectal carcinogenesis. METHODS: Formalin-fixed, paraffin-embedded tissue blocks were obtained. Seven adenocarcinomas and seven adenomas were selected for the presence of mutant c-K-ras genes and histologic transitions between normal and neoplastic tissue. Tissue sections were prepared for analysis by the selective UV radiation fractionation method by placing thin, fixed tissue sections on a plastic slide with no coverslip. Under the microscope, small ink dots from a felt-tip pen were manually placed directly on relatively pure cell subpopulations. The slides were placed with the tissue side exposed to a UV transilluminator for 2-4 hours to inactivate the DNA present in the unprotected ("undotted") cells. Individual dots were cut out of the plastic slide into 2 x 2-mm squares and placed into microfuge tubes. The DNA was extracted and supernatant used for polymerase chain reaction (PCR) analysis. Mutations at c-K-ras codons 12 and 13 were detected. RESULTS: The selective UV radiation fractionation method and PCR analysis revealed that c-K-ras mutations never extended into normal mucosa and were present in all neoplastic cells regardless of phenotypes in all seven adenocarcinomas and three of the seven adenomas. Further examination of two carcinomas for p53 (also known as TP53) mutations or loss of heterozygosity demonstrated that these additional mutations were also present in all tumor cells, suggesting that a single transformed clone was responsible for the majority of growth. However, in four other adenomas, tumor heterogeneity was demonstrated, since c-K-ras mutations were detected only in discrete portions. CONCLUSIONS: Adenoma formation may include a stage in which multiple and genetically distinct neoplastic clones are present, while most carcinomas appear to have a homogeneous composition that may result from the successful progression of one of these clones.

Frameshift somatic mutations in gastrointestinal cancer of the microsatellite mutator phenotype.

An exacerbated genomic instability characterizes hereditary and sporadic gastrointestinal cancer of the microsatellite mutator phenotype (MMP), generating somatic frameshift mutations in genes containing mononucleotide repeats. We have recently shown that approximately 50, 40, and 30% of MMP+ colon tumors harbor frameshift mutations in (G)8, (A)8, and (C)8 tracks within the proapoptotic gene BAX and the hMSH3 and hMSH6 DNA mismatch repair genes, respectively. Here we report a higher incidence of frameshift mutations in these 3 genes in a panel of 25 MMP+ gastric adenocarcinomas: 64% in BAX and hMSH3, and 52% in hMSH6. These results support a multiple mutator gene model for the stepwise unfolding of the MMP and further illustrate the importance of the escape from apoptosis in gastrointestinal cancer. The tumor suppressor role played by BAX is also supported by the finding of other somatic BAX mutations, including recurrent missense mutations, not only in gastrointestinal cancer of the MMP but also in gastrointestinal cancer without the MMP.

Differential expression of the normal and mutated K-ras alleles in chemically induced thymic lymphomas.

The presence of point mutations in the K-ras gene was examined in murine thymic lymphomas induced by a single dose of N-methylnitrosourea by the RNase A mismatch cleavage method and by allelic-specific oligonucleotide hybridization of in vitro amplified DNA by polymerase chain reaction. The results show that the frequency of mutations is lower than that of tumors induced by multiple N-methylnitrosourea treatments. Four mutations identified were the aspartic acid at codon 12, a G:C to A:T transition in its second position. A G:C to T:A transversion in codon 146 was also found in one thymic lymphoma, changing the amino acid alanine to serine. The use of the RNase A assay allowed an estimation of the relative expression levels of both normal and mutant K-ras alleles. The results show that in approximately one half of the tumors the mutant allele is predominantly expressed, suggesting that the normal allele has been lost or that the mutant allele has been amplified relative to the normal. Altogether, these findings are consistent with ras mutations occurring in some instances during tumor development and with a ras effect being not strictly dominant but favoring selection for increasing levels of expression from the oncogenic allele.

Impairment of the proapoptotic activity of Bax by missense mutations found in gastrointestinal cancers.

We have reported previously that codon 169 of the proapoptotic gene BAX is a mutational hot spot in gastrointestinal cancer. Two different mutations were found in this codon, replacing the wild-type threonine by alanine or methionine. To compare the proapoptotic activity of these Bax mutants with wild-type Bax, we established an ecdysone (muristerone A)-inducible system in cultured human embryonal kidney 293 cells. Addition of muristerone A induced a dose-dependent decrease in the viability of cells transfected with wild-type BAX, but this loss of viability was inhibited in cells transfected with BAX mutants. Furthermore, muristerone A induced morphological changes characteristic of apoptosis, including cell shrinkage, rounding, formation of apoptotic bodies, detachment and nuclear condensation and fragmentation, in cells transfected with wild-type BAX. These hallmarks of apoptosis were clearly diminished in cells transfected with BAX mutants. Mutation of threonine 169 did not affect the binding of Bax to Bax, Bcl-2, or Bcl-X(L). These results demonstrate that missense mutations at codon 169 of BAX are functional because they inhibit its apoptotic activity. This is the first report of the functional significance of missense mutations in BAX, or any other proapoptotic member of the Bcl-2 family, in primary human tumors.

Frameshift mutations at mononucleotide repeats in caspase-5 and other target genes in endometrial and gastrointestinal cancer of the microsatellite mutator phenotype.

The majority of tumors from hereditary nonpolyposis colorectal cancer families and a subset of unselected gastrointestinal and endometrial tumors exhibit a microsatellite mutator phenotype (MMP) that leads to the accumulation of hundreds of thousands of clonal mutations in simple repeat sequences. The mutated genes with positive or negative roles in cell growth or survival in aneuploid gastrointestinal cancer (e.g., APC, K-ras, and p53) are less frequently mutated in near-diploid MMP gastrointestinal tumors. These tumors accumulate mutations in other genes, such as DNA mismatch repair hMSH3 and hMSH6, transforming growth factor-beta type II receptor, and BAX. All these genes carry, within their coding sequences, mononucleotide repeats that are preferred targets for the MMP. Endometrial carcinoma is the most common type of extracolonic neoplasia in the hereditary nonpolyposis colorectal cancer syndrome, but the spectrum of its target cancer genes is not well characterized. Here, we report that endometrial cancer of the MMP also accumulates mutations in genes that are typically mutated in gastrointestinal cancer of the mutator pathway, including BAX (55%), hMSH3 (28%), and hMSH6 (17%). We also report the detection of frameshift mutations in caspase-5, a member of the caspase family of proteases that has an (A)10 repeat within its coding region, in MMP tumors of the endometrium, colon, and stomach (28, 62, and 44%, respectively). We therefore suggest caspase-5 as a new target gene in the microsatellite mutator pathway for cancer.

Genetic and clinical features of human pancreatic ductal adenocarcinomas with widespread microsatellite instability.

The incidences of microsatellite instability (MSI) and underlying DNA mismatch repair (MMR) defects in pancreatic carcinogenesis have not been well established. We analyzed 100 sporadic and 3 hereditary pancreatic ductal adenocarcinomas for MSI, and high-frequency MSI (MSI-H) and low-frequency MSI (MSI-L) tumors were further analyzed for frameshift mutations of possible target genes and for promoter methylation and mutation of DNA MMR genes, including hMLH1, hMSH2, hMSH3, and hMSH6 genes. Among the 100 sporadic tumors, 13 (13%) were MSI-H, 13 (13%) were MSI-L, and 74 (74%) were microsatellite stable (MSS) tumors. All of the three hereditary tumors from hereditary nonpolyposis colorectal cancer (HNPCC) patients were MSI-H. MSI-H tumors were significantly associated with poor differentiation and the presence of wild-type K-RAS and p53 genes. Patients with MSI-H tumors had a significantly longer overall survival time than did those with MSI-L or MSS tumors (P = 0.0057). Frameshift mutations of hMSH3, hMLH3, BRCA-2, TGF-beta type II receptor, and BAX genes were detected in MSI-H tumors. Hypermethylation of the hMLH1 promoter was observed in 6 (46%) of the 13 sporadic MSI-H tumors but not in any of the 3 hereditary MSI-H tumors or 13 MSI-L tumors. All of the 3 HNPCC cases had germ-line hMLH1 mutation accompanied by loss of heterogeneity or other mutation in the tumor. Our results suggest that pancreatic carcinomas with MSI-H represent a distinctive oncogenic pathway because they exhibit peculiar clinical, pathological, and molecular characteristics. Our results also suggest the principal involvement of epigenetic or genetic inactivation of the hMLH1 gene in the pathogenesis of pancreatic carcinoma with MSI-H.

Frequent frameshift mutations of RIZ in sporadic gastrointestinal and endometrial carcinomas with microsatellite instability.

Many lines of evidence suggest that the retinoblastoma protein interacting zinc finger gene RIZ is a strong candidate for the tumor suppressor locus on 1p36, a region commonly deleted in many human cancers with chromosomal instability. In addition, a role for RIZ in tumors of the microsatellite instability pathway is suggested by frequent frameshift mutations in hereditary non-polyposis colorectal carcinomas. Here we studied RIZ mutations in sporadic cancers with microsatellite instability. Frameshift mutations in the two coding polyadenosine tracks of RIZ were found in 19 (48%) of 40 gastric carcinomas, 6 (33%) of 18 endometrial carcinomas, 14 (26%) of 51 of colorectal carcinomas, and 7 (54%) of 13 cell lines. Eleven tumor tissues showed biallelic inactivation of RIZ. In contrast, no frameshift mutations were found in 70 microsatellite stable tumors. These results suggest an important role for RIZ in sporadic cancers with microsatellite instability.

Somatic frameshift mutations in DNA mismatch repair and proapoptosis genes in hereditary nonpolyposis colorectal cancer.

An exacerbated genomic instability at simple repeated sequences characterizes cancer of the microsatellite mutator phenotype (MMP). The majority of hereditary nonpolyposis colon cancers (HNPCCs) and about 15% of nonselected ("sporadic") gastrointestinal tumors belong to the MMP pathway of tumorigenesis. Colorectal MMP+ and MMP- tumors exhibit fundamental differences in genotype and phenotype. We have shown previously that "sporadic" MMP+ colon cancers exhibit a paradoxical low incidence of somatic mutations in the p53 tumor suppressor gene and the c-K-ras proto-oncogene. On the other hand, gastrointestinal MMP+ cancers frequently harbor frameshift mutations in genes containing mononucleotide repeats. These include the cell growth regulator gene TGFbetaRII and the proapoptotic gene BAX. We have also recently shown the frequent presence of frameshift mutations in (A)8 and (C)8 tracts within the hMSH3 and hMSH6 DNA mismatch repair genes in sporadic colon cancer of the MMP. Here, we describe the nearly identical incidence of somatic frameshift mutations in these genes in a panel of 27 HNPCC MMP+ cancers: 52% in hMSH3 and BAX and 33% in hMSH6. In contrast, no mutations in any of these genes were found in 10 MMP- cancers of HNPCC patients. These results show that the multistep model for the unfolding of the MMP also applies to HNPCC and further illustrate the importance of the escape from apoptosis in the MMP pathway for gastrointestinal cancer. They also underscore the differences in genotype between tumors with and without enhanced microsatellite instability and the similarities in genotype between tumors of the MMP regardless of their hereditary or sporadic nature.

Detection of c-K-ras mutations in fine needle aspirates from human pancreatic adenocarcinomas.

Formalin-fixed paraffin-embedded tissue specimens obtained by fine needle aspiration of pancreatic masses from 47 patients were examined retrospectively for cytology and the presence of mutant c-K-ras oncogenes. Point mutations of c-K-ras in codon 12 were detected by RNA-DNA RNAse A mismatch cleavage after in vitro DNA amplification of the cellular c-K-ras sequences by the polymerase chain reaction. Of the 36 patients with pancreatic adenocarcinoma, mutant c-K-ras oncogenes were detected in 18 of 25 (72%) with malignant cytologies, 2 of 8 (25%) with atypical cytologies, and 0 of 3 with benign aspiration cytologies. The remaining 11 patients without pancreatic adenocarcinomas did not have mutant c-K-ras genes detectable by the assay. The diagnosis of pancreatic adenocarcinoma was based upon clinical follow-up. The presence of mutant c-K-ras oncogenes did not significantly affect survival in the patients studied. Mutant c-K-ras genes were found at the time of initial clinical presentation in the majority of pancreatic adenocarcinomas, suggesting an important role of the mutation in oncogenesis. In conjunction with cytology, our approach represents an application for cancer diagnosis at the molecular genetic level.

Chromosome 7q allelic losses in pancreatic carcinoma.

During our DNA fingerprinting studies of paired normal and pancreatic cancer tissues using arbitrarily primed PCR, we noticed a band showing an apparent homozygous deletion in a pancreatic cancer cell line and a decreased intensity in a number of primary cancers. That band was assigned to chromosome 7. Such information led us to analyze chromosome 7 loss of heterozygosity (LOH) in a panel of 12 cryostat-enriched primary pancreatic cancers and 2 pancreatic cancer cell lines, despite the reportedly low frequency of chromosome 7 LOH in xenograft-enriched pancreatic cancers. Seventeen PCR-amplified CA-microsatellite polymorphic sites were analyzed. One of the two cell lines and eight common-type cancers (including all five poorly differentiated and three of five moderately differentiated cancers) showed chromosome 7q LOH, whereas the two uncommon types of ductal cancer (one adenosquamous and one mucinous noncystic) scored negative. Our data suggest that chromosome 7q LOH is a frequent event (80%) in cryostat-enrichable common pancreatic ductal carcinomas, that is, those primarily of high cellularity. The chromosome 7q smallest common deleted region described by our cases was between 7q31.1 and 7q32.

Initial characterization of a potential transcriptional enhancer for the human c-K-ras gene.

The nucleotide sequence of the 5' end distal region of the human c-K-ras gene promoter was determined. This region, coincident with a variable DNAse I hypersensitive site in native chromatin, contains sequence similarities with known enhancers. A 400 bp MstII DNA fragment of this region stimulated in cis the correctly initiated transcription of the human beta-globin gene in transfected Hela cells. The stimulation of beta-globin transcription (5-6 fold) was dependent on the distance and orientation of the c-K-ras sequences and on the presence of the CCAAT and CACCC elements in the beta-globin promoter. Interaction of nuclear factors with these c-K-ras sequences was analysed by DNAase I footprinting assays using Hela nuclear extracts. A protein binding to these sequences was identified as nuclear factor 1 (NF-1) by DNAase I competition footprinting experiments. However, disruption of the c-K-ras NF-1 binding site by insertion mutagenesis had no effect on the transcriptional activity of the c-K-ras element.

The microsatellite mutator phenotype of colon cancer cells is often recessive.

A new mutator mechanism for tumorigenesis, characterized by somatic genomic instability (SGI) at simple repeated sequences (SRS) or microsatellites, underlies hereditary nonpolyposis colorectal cancer (HNPCC) and some sporadic tumors of the colon and other types. To determine whether the microsatellite mutator phenotype (MMP) is dominant or recessive, we generate somatic cell hybrids between a tumor cell line without SGI at SRS (D98OR) and colon carcinoma cell lines with relative low (HCT-15) and high (LS174-T) SGI at SRS. The normal fidelity of replication of these unstable sequences was observed in each of these cell hybrids. Fusion of HCT-15/DLD-1 low instability cells, with LS174-T, HCT116 and LoVo cell lines, all exhibiting relative high instability, also restored the replication fidelity of SRS in all of the hybrids. Hybrids between the high instability cell lines did not grow possibly because of senescence or apoptosis. These results indicate that, in the cell lines analysed, the characterized mutator phenotype of the mismatch repair system resulting in high SGI at SRS, and the uncharacterized mutator phenotype underlying low SGI at SRS, are both recessive. The results also suggest that different tumor cells of the MMP harbor distinct altered growth-related genes.

Differential contribution of the ERK and JNK mitogen-activated protein kinase cascades to Ras transformation of HT1080 fibrosarcoma and DLD-1 colon carcinoma cells.

Although an important contribution of ERK and JNK mitogen-activated protein kinase (MAPK) activation in Ras transformation of rodent fibroblasts has been determined, their role in mediating oncogenic Ras transformation of human tumor cells remains to be established. We have utilized the human HT1080 fibrosarcoma and DLD-1 colon carcinoma cell lines, which contain endogenous mutated and oncogenic N- and K-ras alleles, respectively, to address this role. Study of these cells is advantageous over Ras-transformed rodent model cell systems for two key reasons. First, the ras mutations occurred naturally in the progression of the tumors from which the cell lines were derived, rather than due to overexpression of an exogenously introduced gene. Second, although these tumor cells possess defects in multiple genetic loci, it has been established that mutated Ras contributes significantly to the transformed phenotype of these cells. Clonal variant lines of HT1080 and DLD-1 have been isolated which have lost the oncogenic ras allele and exhibit a corresponding impairment in growth transformation in vitro and in vivo. We found that upregulation of Raf/MEK/ERK and JNK correlated with expression of oncogenic Ras in HT1080, but not DLD-1 cells. Furthermore, inhibition of ERK activation in parental HT1080 cells caused the same changes in cell morphology and actin stress fiber organization seen with loss of expression of activated N-Ras(61K). Thus, we suggest that constitutive activation of the Raf/MEK/ERK and JNK pathways is necessary for Ras-induced transformation of HT1080 but not DLD-1 cells. These results emphasize that cell type differences exist in the signaling pathways by which oncogenic Ras causes transformation.

Study of the interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA.

Glyceraldehyde-3-phosphate dehydrogenase binds to homologous and heterologous single-stranded but not double-stranded DNA. Binding to RNA, poly(A) and poly(dA-dT) has also been observed. Enzyme binding to these nucleic acids leads to the formation of an insoluble complex which can be sedimented at low speed. The interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA is strongly inhibited by NAD and NADH but not by NADP. Adenine nucleotides, which inhibit the dehydrogenase activity by competing with NAD for its binding site (Yang, S.T. and Deal, W.C., Jr. (1969) Biochemistry 8, 2806--2813), also inhibit enzyme binding to DNA, whereas glyceraldehyde-3-phosphate and inorganic phosphate are non-inhibitory. These results suggest that DNA interacts through the NAD binding sites of glyceraldehyde-3-phosphate dehydrogenase. In accordance with this idea, it was found that DNA also binds to lactate dehydrogenase, an enzyme containing a similar dinucleotide binding domain, and that this binding is inhibited by NADH. A study of the base specificity of the DNA-glyceraldehyde-3-phosphate dehydrogenase interaction using dinucleoside monophosphates shows that inhibition of DNA binding by the dinucleotides requires the presence of a 3'-terminal adenosine and is greater when the 5'-terminus contains a pyrimidine instead of a purine. These results suggest that the dinucleotides bind at the NAD site of the dehydrogenase and that the enzyme would interact preferentially with PypA dinucleotides present in the nucleic acid.

Assessment of genomic damage in colorectal cancer by DNA fingerprinting: prognostic applications.

PURPOSE: Here we evaluate the prognostic significance of the relative value of genomic damage assessed by DNA fingerprinting in colorectal cancer. MATERIALS AND METHODS: Sixty-three tumor and paired normal mucosa samples were included in the study. Genomic damage was assessed by comparative analysis of paired normal and tumor tissue DNA fingerprints by the arbitrarily primed polymerase chain reaction (AP-PCR). Decreases and increases of intensity in bands were computed and referred to the total number of visualized bands per case. An index reflecting the genomic damage fraction (GDF), with separated values for losses and gains, was obtained for each tumor. This index was used to determine molecular and clinicopathologic correlates after exclusion of eight cases displaying microsatellite instability. RESULTS: Fifty-five cases were considered for the statistical analysis. The average fraction of altered bands per tumor was 0.287+/-0.121. When losses and gains were computed separately, the average fraction of changes was 0.126+/-0.113 and 0.161+/-0.120, respectively. Tumors lacking a ras mutation showed an increased GDF, primarily because of a higher fraction of gains. Tumors that were at advanced Dukes' stages and that were poorly differentiated also displayed a higher GDF. Finally, disease-free survival was significantly diminished in tumors with a GDF greater than 0.314 (P < .001). The prognostic significance of the GDF was independent of Dukes' stage (Cox multivariate analysis, P = .005). CONCLUSION: The degree of genomic damage assessed by unbiased DNA fingerprinting correlates with genotypic, phenotypic, and clinical variables in colorectal carcinoma and may be useful in assessing prognosis in colorectal cancer.